Many biological organisms trigger allergic reactions, respiratory difficulties, and some types of asthma. Some molds release mycotoxins and metabolites that may cause respiratory or thermal discomfort. These spores are the reproductive units of fungi and their elimination has become a matter of importance in enhancing the general well-being of man.
A discussion of conventional techniques for microscopic examination of growth such as fungi is set forth at pages 180-181 of “Medically Important Fungi—A Guide to Identification” by Davise H. Larone (1987). Three most popular types of microscopic examination techniques include tease-mount which is a rapid method but is destructive and does not always preserve the original position and structure of the conidia, spores and the like, both of which are extremely important to allow accurate identification. Cellophane tape-mount is also a rapid method and uses tape to remove part of the growth before the fungi is placed on a small drop of LPCB on a glass slide and examined under a microscope. This method helps retain the original position of characteristic fungal structures. Slide culture techniques preserve the structure of a fungus in a slide culture; however these is not as rapid as other techniques since they require suspending a microscopic slide in a petri dish with a block of agar on top of the slide. Once the fungi have grown to a stage sufficient to permit proper identification, a slide culture is generally made from this initial culture. The slide culture generally consists of a square of agar medium which has been inoculated with a sample of the culture of the fungi initially made, and which has been placed upon a microscope slide with a cover glass on top of the culture on the slide. The slide culture is then generally incubated in a humid environment. After incubation, identification of the fungi is generally accomplished by removing the cover glass from the slide, placing the cover glass on another microscope slide on top of a drop of lacto-phenol cotton blue stain, and viewing the fungi under a microscope.
A problem with microscopic techniques is that they are dependent upon the individual expertise of the operator, thereby increasing the margin of error both in the over identification of fungi-false positives, and in the under identification of fungi-false negatives whenever the analysis is made by any person other than a certified mycologist.
U.S. Pat. No. 5,839,445 discloses a method of diagnosing a disease of a fingernail, a toenail, skin, or a mucus membrane using a confocal microscope to observe abnormal structures within the fingernail, the toenail, the skin, or the mucus membrane. The characteristics of those structures are compared to corresponding characteristic structures known to be associated with a given disease state. This method includes monitoring the progress of a treatment of a disease using a confocal microscope to observe changes over time of an infected area.
One recent approach to ease these problems, described in U.S. Pat. No. 5,770,441, permits the growth of a culture of microorganisms, such as fungi, in a closed system so that the microorganisms may be viewed at any time under a microscope through a slide system apparatus without interrupting the growth of the microorganisms. This enables an operator working with the culture to view the growing fungi (or other microorganisms) on a daily basis without disturbing the growing fungi in any manner until the fungi have grown to a stage where they can be properly identified; i. e., until they have formed the reproduction bodies known as conidia, which will permit their proper identification and diagnosis. At this stage, the operator can properly identify and diagnose the fungi by placing the slide system apparatus on a microscope, and viewing the growing fungi through the slide system apparatus. This approach is exemplary of conventional methods for identifying microorganisms but suffers the disadvantage of being very time-consuming.
Unfortunately, the rising demand for diagnosing fungal pathogens has exacerbated this situation and tremendous demands are placed on a limited availability of board certified dermatologist. Further, it is becoming important for businesses considering capital expansion to new property or certifying an existing work area to be free of fungal pathogen. Generally, it is best to examine a fungus microscopically when the culture first begins to grow and forms conidia or spores, and again a few days later. In many instances, the manner of conidiation or sporulation, which is critical to the correct identification of the fungi, is obscured in old cultures. Further, many fungi are polymorphic and, therefore, observation of the fruiting bodies as they grow is essential. Such observation is not possible in-situ with currently-existing methods for growing cultures of fungi.
U.S. Pat. No. 6,005,964 discloses a system for automatically detecting the presence of contaminants in samples. The system includes a microscope, a controllable stage positioner for holding slides under the microscope, a computer for controlling the stage positioner and a digital camera to capture images through the microscope. The system scans microscope views of regions of a slide sample and provides the digital images to a computer. Image processing routines stored in the computer analyze the digital images and determine whether these images may contain certain contaminants by comparing the characteristics of the objects in the image with the known characteristics of the contaminants.
U.S. Pat. No. 5,859,700 discloses a digital imaging spectrophotometer configured for the direct determination of the ground state absorption spectra of microscopic samples from approximately 400 nm to approximately 950 nm at 2 nm resolution with approximately 1 micron refraction limited spatial resolution. The instrument also uses image processing techniques to identify features, such as a single microbial cell, on a microscope slide or thin section.
It can therefore be seen that available examination techniques sacrifice accuracy of identification for speed or speed for accuracy of identification. Furthermore, the most accurate method, the slide culture method, does not allow in situ viewing.
American Journal of Clinical Pathology 111 (6), p 792-795 June, 1999, defines telepathology as the practice of pathology at a distance using video-microscopy and telecommunication tools. It mentions the use of “virtual microscopy” techniques and the Internet as tools for tele-pathology gastrointestinal biopsy consultations. Gastrointestinal biopsy specimens were photographed using a high-resolution digital camera, a light microscope, and a microcomputer. Digital photomicrographs collected at 40× or 100× optical magnification using 2,700×3,400 pixel resolution were saved in a JPEG image format. Such images could be magnified digitally up to 600% without visible degradation and scrolled at different magnifications on a video monitor, simulating examination under a light microscope. The image files (281 to 3,324 KB) were attached to e-mail messages containing patient information and sent through the Internet to Michigan for interpretation using a Macintosh™ computer.
Mycopathologia Journal 118 (2), 1992, 65-69, describes a system for the evaluation of antifungal activity of volatile compounds based on dynamic growth of a single hypha. The system is composed of a reaction vessel under a microscope, an automatic stage, a charge coupled device camera, a TV monitor, a video tape recorder, and a microcomputer. A fungus was inoculated in the reaction vessel containing agar medium and then was treated with an antifungal reagent in the gas phase either in batch or flow reaction manner. The apex of a growing hypha displayed on the TV monitor was followed automatically. From the ratio of the growth rate under exposure of a reagent (UBXPO) to the growth rate before the exposure, the antifungal activity was expressed quantitatively.
A solution to the shortcomings of existing systems has heretofore been impeded because of the lack of sufficiently accurate microscopic techniques that would provide for on-site examinations for fungal pathogens enabling board certified dermatologist to make fungi examinations on-site at remote locations.